Recently we have developed a new class of peptide nucleic acid called GPNA that is readily taken up by both human somatic and embryonic stem cells, and binds sequence specifically to DNA and RNA. GPNA designed to bind to the transcription and translation start site of the gene transcript elicited potent antisense effect with unusually low cytotoxicity compared to the unmodified PNA and its other derivatives. The proposed research will explore the scope of this particular class of molecules and establish a basic understanding of the factors and mechanisms that control cellular uptake, hybridization and cytotoxicity over a wide range of cell lines, including human ES cells. Specifically, we aim to accomplish four specific objectives within the next five years. Aim 1 (Section D.1): Evaluate the antisense effects of GPNA with human somatic and ES cells;Aim 2 (Section D.2): Optimize cellular uptake, hybridization and cytotoxicity. Aim 3 (Section D.3): Determine the mechanism of GPNA uptake and cytotoxicity. And Aim 4 (Section D.4): Determine the scope of GPNA in regulating gene expression. The proposed study is crucial to the future design and development of nucleic acid mimics for a safe and effective use in animals and in humans. GPNA is, to the best of our knowledge, the first example of nucleic acid analogue taken up by human ES cells. This is intriguing because these primitive cells are extremely difficult to penetrate and are sensitive to the environmental cues. Human ES cells hold the key to understanding early human development that can neither be studied directly in utero nor fully understood with animals model, not to mention its enormous potential for regenerative medicine. Many fascinating questions concerning human ES cell proliferation, differentiation, cellular lifespan and so forth have not yet been addressed. Furthermore, recent evidence suggests that cancers may arise from stem cells. If this proves to be correct, it will alter the course of cancer treatment. Before we can begin to address these questions, an effective method must be developed to regulate gene expression in these and related cell types - this is the aim of our research.